Stable compositions of high-concentration allotype-selected antibodies for small-volume administration

ABSTRACT

Disclosed are methods, compositions and uses of high concentration antibody or immunoglobulin formulations for subcutaneous, intramuscular, transdermal or other local (regional) administration, in a volume of than 3, less than 2 or less than 1 ml. Preferably, the formulation contains a high concentration formulation (HCF) buffer comprising phosphate, citrate, polysorbate 80 and mannitol at a pH of about 5.2. The formulation more preferably comprises at least 100, 150, 200, 250 mg/ml or 300 mg/ml of antibody. The methods for preparing the high concentration formulation include ultrafiltration and diafiltration to concentrate the antibody and exchange the medium for HCF buffer. Other embodiments concern use of non-G1m1 (nG1m1) allotype antibodies, such as G1m3 and/or a nG1m1,2 antibodies. The nG1m1 antibodies show decreased immunogenicity compared to G1m1 antibodies.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/876,200, filed Oct. 6, 2015, which was a continuation of U.S. patentapplication Ser. No. 14/163,443 (now U.S. Pat. No. 9,180,205), filedJan. 24, 2014, which was a divisional of U.S. patent application Ser.No. 14/132,549 (now U.S. Pat. No. 9,468,689), filed Dec. 18, 2013, whichwas a divisional of U.S. patent application Ser. No. 13/461,307 (nowU.S. Pat. No. 8,658,773), filed May 1, 2012, which claimed the benefitunder 35 U.S.C. 119(e) of provisional U.S. Patent Application Ser. Nos.61/481,489, filed May 2, 2011, and 61/509,850, filed Jul. 20, 2011.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 27, 2012, isnamed IMM332US.txt and is 26,473 bytes in size.

FIELD OF THE INVENTION

The present invention concerns compositions and methods of productionand use of stable, highly concentrated formulations of immunoglobulins,antibodies and/or antigen-binding antibody fragments. In preferredembodiments, the immunoglobulins, antibodies or fragments thereof may benon-specific human immunoglobulin (e.g., IVIg), antibodies that bind toantigens such as CD22 (e.g., epratuzumab), CD20 (e.g., veltuzumab;GA101), CD74 (e.g., milatuzumab) or HLA-DR (e.g., IMMU-114 or hL243),although antibodies against other antigenic targets may be utilized. Inmore preferred embodiments, the allotypes of the antibodies or fragmentsthereof are selected to be non-G1m1 human allotypes, such as G1m3. Evenmore preferred is the nG1m1,2 null allotype. The antibodies or fragmentsthereof may be either naked (unconjugated) or may be conjugated to atleast one therapeutic or diagnostic agent that is not a cytotoxic agentor a radionuclide. The concentrated antibody formulations are of use forthe treatment of a variety of diseases, such as autoimmune disease,immune dysregulation disease, infectious disease, cancer (e.g.,carcinoma, sarcoma, melanoma, glioma, neuroblastoma, lymphoma, leukemia,chronic lymphocytic leukemia, follicular lymphoma, diffused large B-celllymphoma, T-cell lymphoma or leukemia, multiple myeloma or non-Hodgkin'slymphoma), cardiovascular, neurological or metabolic disease. In otherpreferred embodiments, the concentrated antibody formulations may beadministered by subcutaneous, intramuscular or transdermaladministration, although other modes of administration (e.g.,intrathecal, intraperitoneal, intraocular, intracranial, etc.) arecontemplated. In most preferred embodiments, the antibodies or fragmentsthereof are concentrated to at least 80 mg/ml, more preferably at least100 mg/ml, more preferably at least 125 mg/ml, more preferably at least150 mg/ml, more preferably at least 175 mg/ml, more preferably at least200 mg/ml, more preferably at least 225 mg/ml, more preferably at least250 mg/ml, more preferably at least 300 mg/ml in a slightly acidicaqueous buffer solution. Other components of the formulation may includebuffers, such as citrate or phosphate, salts such as sodium chloride,surfactants such as polysorbate 80 and/or polyols such as mannitol.

BACKGROUND

Administration of monoclonal antibodies or fragments thereof has beenproposed for diagnosis and/or therapy of a wide variety of diseasestates, such as cancer, infectious diseases, autoimmune or immunedysfunction disease, neurological diseases, cardiovascular disease andmetabolic disease. (See, e.g., Nadler et al., 1980, Cancer Res40:3147-54; Ritz and Schlossman, 1982, Blood 59:1-11; Waldmann, 2003,Nature Med 9:269-77; Ibbotson et al., 2003, Am J Cardiovasc Drugs3:381-86; Dorner et al., 2009, Nat Rev Rheumatol 5:433-41; Pul et al.,2011, Expert Opin Biol Ther 11:343-57). Human immunoglobulin mixturesare also used, particularly by subcutaneous injection, for the treatmentof hepatitis, as well as various autoimmune diseases by intravenousinfusion (see, e.g., Powell et al., 2006, Clin Transplant 20:524-25;Stiehm, 1997, Pediatr Infect Dis J 16:696-707; Zandman et al., Clin RevAllergy Immunol [Epub ahead of print, Jul. 6, 2011]; Kaveri et al.,2011, Clin Exp Immunol 164:2-5).

While intravenous infusion has been the standard mode of antibodyadministration, infusion-related reactions such as rash, urticaria,erythema, pruritus, hypotension, bronchospasm or anaphylaxis may besevere and can significantly limit the rate of antibody infusion. (See,e.g., Kang and Saif, 2007, J Supportive Oncol 5:451-57; Vogel, 2010,Clin J Oncol Nursing 14:E10-21). In part to address the incidence ofinfusion-related reactions, subcutaneous administration of therapeuticantibodies has been proposed (Lundin et al., 2002, Blood 100:768-73;Kavanaugh et al., Arthritis Rheum, 2009, 60:976-86; Negrea et al. 2011,Haematologica 96:567-73). Intramuscular administration is also given,such as with IVIg (Marzano et al., 2010, Minerva Med 101:373-83;Pauwelyn et al., 2010, Transplant Proc 42:4399-402; Filipponi et al.,2010, Dig Liver Dis 42:509-14). Another alternative is transdermaladministration (e.g., Burton et al., 2011, Pharm Res 28:31-40; Wendorfet al., 2011, Pharm Res 28:22-30; Koutsonanos et al., 2009, PLoS One4:e4773). While infusion-site reactions may still occur, subcutaneous,intramuscular or transdermal administration would result in decreasedhealth care costs by avoiding the need for lengthy intravenousadministration and dedicated infusion suites and staff, and may alsodecrease the incidence of systemic infusion reactions (Lundin et al.,2002, Blood 100:768-73; Wasserman, 2008, Patient Preference andAdherence, 2:163-66; Negrea et al. 2011, Haematologica 96:567-73), aswell as being more tolerable and convenient for the patient, includingthe possibility for self-administration. Because of the lower injectionvolume associated with subcutaneous, intramuscular or transdermaladministration, a need exists for more concentrated antibody orimmunoglobulin formulations that are stable for long periods of time andcan be administered subcutaneously, intramuscularly or transdermally (orby other routes requiring small volumes of injectate).

SUMMARY

The present invention concerns compositions and methods of productionand use of stable, highly concentrated formulations of therapeuticimmunoglobulins, monoclonal antibodies or antigen-binding fragmentsthereof and use for low-volume injections. Although many methods ofantibody production are known in the art and may be utilized, preferablyan expression vector(s) encoding the antibody or fragment is transfectedinto a mammalian cell line such as SpEEE, SpESF or SpESF-X (see, e.g.,U.S. Pat. Nos. 7,531,327; 7,537,930; 7,608,425; and 7,785,880; theExamples section of each of which is incorporated herein by reference).More preferably, both transfection and antibody expression occur inserum-free medium to decrease the expense of production and remove asource of contaminating proteins. The antibody is produced into the cellculture medium for further purification.

In other preferred embodiments, the antibody may be purified from cellculture medium by sequential chromatography, for example by affinity andion exchange column chromatography. Non-limiting examples includeaffinity chromatography on Protein A, anion-exchange chromatography onQ-SEPHAROSE® and cation-exchange chromatography on SP-SEPHAROSE®. Morepreferably, the antibody is bound to the SP-SEPHAROSE® resin in pH 5citrate buffer and eluted from the column with pH 6 citrate buffer in0.15 M NaCl. The eluate from the SP-SEPHAROSE® column may be filteredthrough, for example, a 20 nm filter for virus removal. The purifiedantibody may then be diafiltered, for example using an AMICON®Ultrafiltration Cell with a 50 KD MW cut-off filter to exchange themedium with a high concentration formulation buffer (HCF buffer) and toconcentrate the antibody for storage. In most preferred embodiments, theHCF buffer solution may comprise phosphate buffer (pH 5.2), sodiumchloride, Polysorbate 80, citrate and mannitol. Polysorbate 80 serves todecrease protein aggregation, while mannitol stabilizes the antibody inaqueous medium. The diafiltration concentrates the antibody topreferably at least 80 mg/ml, more preferably at least 100 mg/ml, morepreferably at least 150 mg/ml, more preferably at least 200 mg/ml, morepreferably at least 300 mg/ml final concentration. The concentratedantibody exhibits little or no aggregation and preferably is stable inliquid form at 2-8° for at least 10 months. In even more preferredembodiments, the Polysorbate 80 is added to the concentrated antibodyafter the ultrafiltration step.

The stable, highly concentrated antibody is of use for preparingmedicaments for administration to subjects, preferably by subcutaneous,transdermal or intramuscular administration. However, the skilledartisan will realize that other forms of administration known in theart, such as intravenous, intraperitoneal, intraventricular,intraocular, and/or intrathecal administration may be utilized.

Antibodies of use may bind to any disease-associated antigen known inthe art. Where the disease state is cancer, for example, many antigensexpressed by or otherwise associated with tumor cells are known in theart, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein, α-actinin-4, A3, antigen specific for A33 antibody,ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m,CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132,CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CXCR4, CXCR7,CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6,c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3,folate receptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1(IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophagemigration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2,NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3,MUC4, MUC5, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostaticacid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25,RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72,tenascin, TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TROP-2, VEGFR, ED-B fibronectin,WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, anangiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene marker and anoncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CD74, CD20, CD22 or HLA-DR.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. More preferably, the antibody is hA20 (veltuzumab),hLL2 (epratuzumab), hLL1 (milatuzumab) or hL243 (IMMU-114).

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20),tositumomab (anti-CD20), trastuzumab (anti-ErbB2), abagovomab(anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6receptor), benralizumab (anti-CD125), CC49 (anti-TAG-72), AB-PG1-XG1-026(anti-PSMA, U.S. patent application Ser. No. 11/983,372, deposited asATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575),tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab(anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20; Glycart Roche),muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 integrin),omalizumab (anti-IgE); anti-TNF-α antibodies such as CDP571 (Ofei etal., 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI,M302B, M303 (Thermo Scientific, Rockford, Ill.), infliximab (Centocor,Malvern, Pa.), certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L(UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park, Ill.),Benlysta (Human Genome Sciences); antibodies for therapy of Alzheimer'sdisease such as Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem263:7943-47), gantenerumab, solanezumab and infliximab; anti-fibrinantibodies like 59D8, T2G1s, MH1; anti-HIV antibodies such as P4/D10(U.S. patent application Ser. No. 11/745,692), Ab 75, Ab 76, Ab 77(Paulik et al., 1999, Biochem Pharmacol 58:1781-90); and antibodiesagainst pathogens such as CR6261 (anti-influenza), exbivirumab(anti-hepatitis B), felvizumab (anti-respiratory syncytial virus),foravirumab (anti-rabies virus), motavizumab (anti-respiratory syncytialvirus), palivizumab (anti-respiratory syncytial virus), panobacumab(anti-Pseudomonas), rafivirumab (anti-rabies virus), regavirumab(anti-cytomegalovirus), sevirumab (anti-cytomegalovirus), tivirumab(anti-hepatitis B), and urtoxazumab (anti-E. coli).

An antibody or antigen-binding fragment of use may be chimeric,humanized or human. The use of chimeric antibodies is preferred to theparent murine antibodies because they possess human antibody constantregion sequences and therefore do not elicit as strong a humananti-mouse antibody (HAMA) response as murine antibodies. The use ofhumanized antibodies is even more preferred, in order to further reducethe possibility of inducing a HAMA reaction. Techniques for humanizationof murine antibodies by replacing murine framework and constant regionsequences with corresponding human antibody framework and constantregion sequences are well known in the art and have been applied tonumerous murine anti-cancer antibodies. Antibody humanization may alsoinvolve the substitution of one or more human framework amino acidresidues with the corresponding residues from the parent murineframework region sequences. As discussed below, techniques forproduction of human antibodies are also well known.

The therapeutic formulation may comprise an antibody fragment, such asF(ab′)₂, Fab, scFv, Fv, or a fusion protein utilizing part or all of thelight and heavy chains of the F(ab′)₂, Fab, scFv. The antibody may alsobe multivalent, or multivalent and multispecific. The antibody mayinclude human constant regions of IgG1, IgG2a, IgG3, or IgG4.

In more preferred embodiments, the allotype of the antibody may beselected to minimize host immunogenic response to the administeredantibody, as discussed in more detail below. A preferred allotype is anon-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. Thenon-G1m1 allotype is preferred for decreased antibody immunoreactivity.Surprisingly, repeated subcutaneous administration of concentrated nG1m1antibody was not found to induce significant immune response, despitethe enhanced immunogenicity of subcutaneous administration.

Various embodiments may concern use of the subject methods andcompositions to treat a disease, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute andchronic myeloid leukemias, T-cell lymphomas and leukemias, multiplemyeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas, melanomas,sarcomas, gliomas, and skin cancers. The carcinomas may includecarcinomas of the oral cavity, gastrointestinal tract, pulmonary tract,lung, breast, ovary, prostate, uterus, endometrium, cervix, urinarybladder, pancreas, bone, brain, connective tissue, liver, gall bladder,kidney, skin, central nervous system, and testes.

In addition, the subject methods and compositions may be used to treatan autoimmune disease, for example acute immune thrombocytopenia,chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, pemphigusvulgaris, diabetes mellitus (e.g., juvenile diabetes), Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, or fibrosing alveolitis.

In alternative embodiments, the concentrated antibody formulation may beof use to treat a metabolic disease, such as type-2 diabetes oramyloidosis, a cardiovascular disease, such as atherosclerosis, or aneurologic disease, such as Alzheimer's disease. Antibodies of use fortherapy of such conditions are known in the art, as discussed in moredetail below.

In certain embodiments, disease therapy may be enhanced by combinationtherapy with one or more other therapeutic agents as part of thisinvention. Known therapeutic agents of use in this invention includeimmunomodulators (such as cytokines, lymphokines, chemokines, and growthfactors, and their inhibitors), sphingosine inhibitors, hormones,hormone antagonists, oligonucleotides (such as siRNA or RNAi),photoactive therapeutic agents, anti-angiogenic agents and pro-apoptoticagents. Other more traditional therapeutic agents, such as cytotoxicdrugs or radionuclides, may be administered before, concurrently with,or after the concentrated antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate preferred embodimentsof the invention. However, the claimed subject matter is in no waylimited by the illustrative embodiments disclosed in the drawings.

FIG. 1. Exemplary protocol for column chromatography purification ofantibody from cell culture medium.

FIG. 2. SDS-polyacrylamide gel electrophoresis of ultrafiltrationconcentrated antibodies: (A) non-reducing gel, (B) reducing gel. Bothgels show (lane 1) MW standards; (lane 2) hLL1 IgG, starting IgGsolution (10 mg/mL); (lane 3) concentrated hLL1 IgG, after 2 monthstorage, (215 mg/mL); (lane 4) hA20 IgG, starting IgG solution (5.1mg/mL); (lane 5) concentrated hA20 IgG, after 10 month storage, (162mg/mL); (lane 6) hL243 IgG, starting IgG solution (8.9 mg/mL); (lane 7)concentrated hL243 IgG, after 10 month storage, (101 mg/mL). The MWstandards used were respectively 6.5, 14, 21, 31, 45, 66, 97, 116 and200 KD.

FIG. 3. Isoelectric focusing gel of ultrafiltration concentratedantibodies showing (lane 1) pI standards; (lane 2) hLL1 IgG, startingIgG solution (10 mg/mL); (lane 3) concentrated hLL1 IgG, after 2 monthstorage, (215 mg/mL); (lane 4) hA20 IgG, starting IgG solution (5.1mg/mL); (lane 5) concentrated hA20 IgG, after 10 month storage, (162mg/mL); (lane 6) hL243 IgG, starting IgG solution (8.9 mg/mL); (lane 7)concentrated hL243 IgG, after 10 month storage, (101 mg/mL). The MWstandards used were respectively 6.5, 14, 21, 31, 45, 66, 97, 116 and200 KD.

FIG. 4. Representative SE HPLC chromatogram of ultrafiltrationconcentrated hLL1 IgG solution (215 mg/mL) after 10 months of storage.

FIG. 5. Representative SE HPLC chromatogram of ultrafiltrationconcentrated hA20 IgG solution (162 mg/mL) after 10 months of storage.

FIG. 6. Representative SE HPLC chromatogram of ultrafiltrationconcentrated hL243 IgG solution (101 mg/mL) after 10 months of storage.

FIG. 7. Comparison of veltuzumab (SEQ ID NO:33) vs. rituximab (SEQ IDNO:34) heavy chain constant region sequences. Identical residues areindicated by asterisks. The two different allotype antibodies differ inheavy chain constant region sequence by only four amino acid residues.The light chain constant region sequences are identical between the twoantibodies.

FIG. 8. The amino acid sequences of hL243. The entire variable andconstant region sequence of hL243 IgG4P heavy chain is shown as SEQ IDNO:37, with the constant region underlined. The constant region sequencealone of hL243 IgG4P heavy chain is shown as SEQ ID NO:38. The entirevariable and constant region sequence of the hL243 light chain is shownas SEQ ID NO:39, with the constant region underlined. The constantregion sequence alone of hL243 light chain is shown as SEQ ID NO:40.

DETAILED DESCRIPTION Definitions

The following definitions are provided to facilitate understanding ofthe disclosure herein. Where a term is not specifically defined, it isused in accordance with its plain and ordinary meaning.

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

An “antibody” refers to a full-length (i.e., naturally occurring orformed by normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., antigen-binding) portion of an immunoglobulin molecule,like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, single domain antibodies (DABs or VHHs) andthe like, including half-molecules of IgG4 (van der Neut Kolfschoten etal., 2007, Science 317:1554-1557). Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-CD74 antibody fragment binds with anepitope of CD74. The term “antibody fragment” also includes isolatedfragments consisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains, including human framework region(FR) sequences. The constant domains of the antibody molecule arederived from those of a human antibody.

A “human antibody” is an antibody obtained from transgenic mice thathave been genetically engineered to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) forthe production of human antibodies and fragments thereof in vitro, fromimmunoglobulin variable domain gene repertoires from unimmunizeddonors). In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see, e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.(See, U.S. Pat. Nos. 5,567,610 and 5,229,275).

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includebut are not limited to antibodies, antibody fragments, drugs, cytokineor chemokine inhibitors, proapoptotic agents, tyrosine kinaseinhibitors, toxins, enzymes, nucleases, hormones, immunomodulators,antisense oligonucleotides, siRNA, RNAi, chelators, boron compounds,photoactive agents, dyes and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, fluorescent compoundsor molecules and enhancing agents (e.g., paramagnetic ions). Preferably,the diagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents, and fluorescent compounds.

An “immunoconjugate” is a conjugate of an antibody with an atom,molecule, or a higher-ordered structure (e.g., with a liposome), atherapeutic agent, or a diagnostic agent. A “naked antibody” is anantibody that is not conjugated to any other agent.

A “naked antibody” is generally an entire antibody that is notconjugated to a therapeutic agent. This is so because the Fc portion ofthe antibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity) that setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric, humanized or humanantibodies.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which an antibody or antibodyfragment is linked to another protein or peptide, such as the same ordifferent antibody or antibody fragment or a DDD or AD peptide. Thefusion protein may comprise a single antibody component, a multivalentor multispecific combination of different antibody components ormultiple copies of the same antibody component. The fusion protein mayadditionally comprise an antibody or an antibody fragment and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators and toxins. One preferred toxincomprises a ribonuclease (RNase), preferably a recombinant RNase.

A “multispecific antibody” is an antibody that can bind simultaneouslyto at least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. A “multivalent antibody” is anantibody that can bind simultaneously to at least two targets that areof the same or different structure. Valency indicates how many bindingarms or sites the antibody has to a single antigen or epitope; i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyis able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Multispecific, multivalent antibodiesare constructs that have more than one binding site of differentspecificity.

A “bispecific antibody” is an antibody that can bind simultaneously totwo targets which are of different structure. Bispecific antibodies(bsAb) and bispecific antibody fragments (bsFab) may have at least onearm that specifically binds to, for example, a B cell, T cell, myeloid-,plasma-, and mast-cell antigen or epitope and at least one other armthat specifically binds to a targetable conjugate that bears atherapeutic or diagnostic agent. A variety of bispecific antibodies canbe produced using molecular engineering.

Preparation of Monoclonal Antibodies

The compositions, formulations and methods described herein may includemonoclonal antibodies. Rodent monoclonal antibodies to specific antigensmay be obtained by methods known to those skilled in the art. (See,e.g., Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991)). General techniques for cloning murineimmunoglobulin variable domains have been disclosed, for example, by thepublication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833(1989).

Chimeric Antibodies

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Techniques forconstructing chimeric antibodies are well known to those of skill in theart. As an example, Leung et al., Hybridoma 13:469 (1994), disclose howthey produced an LL2 chimera by combining DNA sequences encoding theV_(k) and V_(H) domains of LL2 monoclonal antibody, an anti-CD22antibody, with respective human and IgG₁ constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(k) and V_(H), respectively.

Humanized Antibodies

A chimeric monoclonal antibody can be humanized by replacing thesequences of the murine FR in the variable domains of the chimericantibody with one or more different human FR. Specifically, mouse CDRsare transferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. (See, e.g., Tempest et al., Biotechnology 9:266 (1991)and Verhoeyen et al., Science 239: 1534 (1988)). Techniques forproducing humanized antibodies are disclosed, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993).

Human Antibodies

A fully human antibody can be obtained from a transgenic non-humananimal. (See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997;U.S. Pat. No. 5,633,425.) Methods for producing fully human antibodiesusing either combinatorial approaches or transgenic animals transformedwith human immunoglobulin loci are known in the art (e.g., Mancini etal., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr.Opin. Pharmacol. 3:544-50; each incorporated herein by reference). Suchfully human antibodies are expected to exhibit even fewer side effectsthan chimeric or humanized antibodies and to function in vivo asessentially endogenous human antibodies. In certain embodiments, theclaimed methods and procedures may utilize human antibodies produced bysuch techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22, incorporated herein by reference). The final Fabfragments were digested with restriction endonucleases and inserted intothe bacteriophage genome to make the phage display library. Suchlibraries may be screened by standard phage display methods. The skilledartisan will realize that this technique is exemplary only and any knownmethod for making and screening human antibodies or antibody fragmentsby phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XenoMouse® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the Vκ andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown in FIG.7 for the exemplary antibodies rituximab (SEQ ID NO:34) and veltuzumab(SEQ ID NO:33).

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1 m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1 and FIG. 7, rituximab(G1m17,1) is a DEL allotype IgG1, with an additional sequence variationat Kabat position 214 (heavy chain CH1) of lysine in rituximab vs.arginine in veltuzumab. It has been reported that veltuzumab is lessimmunogenic in subjects than rituximab (see, e.g., Morchhauser et al.,2009, J Clin Oncol 27:3346-53; Goldenberg et al., 2009, Blood113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), an effect that hasbeen attributed to the difference between humanized and chimericantibodies. However, the difference in allotypes between the EEM and DELallotypes likely also accounts for the lower immunogenicity ofveltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes 214 356/358 431 Complete allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan(anti-CD20); panitumumab (anti-EGFR); rituximab (anti-CD20); tositumomab(anti-CD20); GA101 (anti-CD20); and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865; the Examples section ofeach incorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19(hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al.,2011, Nature Med 17:610-18). Anti-CD3 antibodies also have been proposedfor therapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

The pharmaceutical composition of the present invention may be used totreat a subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease. Bapineuzumab isin clinical trials for Alzheimer's disease therapy. Other antibodiesproposed for therapy of Alzheimer's disease include Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition.

In a preferred embodiment, diseases that may be treated using theclaimed compositions and methods include cardiovascular diseases, suchas fibrin clots, atherosclerosis, myocardial ischemia and infarction.Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) are known and inclinical trials as imaging agents for disclosing said clots andpulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL), anti-MIF (e.g., U.S. Pat. Nos. 6,645,493; 7,517,523,the Examples section of each incorporated herein by reference), andanti-CD74 (e.g., hLL1) antibodies can be used to target atheroscleroticplaques. Abciximab (anti-glycoprotein IIb/IIIa) has been approved foradjuvant use for prevention of restenosis in percutaneous coronaryinterventions and the treatment of unstable angina (Waldmann et al.,2000, Hematol 1:394-408). Anti-CD3 antibodies have been reported toreduce development and progression of atherosclerosis (Steffens et al.,2006, Circulation 114:1977-84). Antibodies against oxidized LDL induceda regression of established atherosclerosis in a mouse model (Ginsberg,2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown toreduce ischemic cell damage after cerebral artery occlusion in rats(Zhang et al., 1994, Neurology 44:1747-51). Commercially availablemonoclonal antibodies to leukocyte antigens are represented by: OKTanti-T-cell monoclonal antibodies (available from Ortho PharmaceuticalCompany) which bind to normal T-lymphocytes; the monoclonal antibodiesproduced by the hybridomas having the ATCC accession numbers HB44, HB55,HB12, HB78 and HB2; G7Ell, W8E7, NKP15 and GO22 (Becton Dickinson);NEN9.4 (New England Nuclear); and FMCll (Sera Labs). A description ofantibodies against fibrin and platelet antigens is contained in Knight,Semin. Nucl. Med., 20:52-67 (1990).

Other antibodies that may be used include antibodies against infectiousdisease agents, such as bacteria, viruses, mycoplasms or otherpathogens. Many antibodies against such infectious agents are known inthe art and any such known antibody may be used in the claimed methodsand compositions. For example, antibodies against the gp120 glycoproteinantigen of human immunodeficiency virus I (HIV-1) are known, and certainof such antibodies can have an immunoprotective role in humans. See,e.g., Rossi et al., Proc. Natl. Acad. Sci. USA. 86:8055-8058, 1990.Known anti-HIV antibodies include the anti-envelope antibody describedby Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference. Antibodies against hepatitis virus are also known and may beutilized (e.g., Dagan and Eren, Curr Opin Mol Ther, 2003, 5:148-55; Kecket al., 2008, Curr Top Microbiol Immunol 317:1-38; El-Awady et al.,2006, 12:2530-35).

Antibodies against malaria parasites can be directed against thesporozoite, merozoite, schizont and gametocyte stages. Monoclonalantibodies have been generated against sporozoites (cirumsporozoiteantigen), and have been shown to neutralize sporozoites in vitro and inrodents (N. Yoshida et al., Science 207:71-73, 1980). Several groupshave developed antibodies to T. gondii, the protozoan parasite involvedin toxoplasmosis (Kasper et al., J. Immunol. 129:1694-1699, 1982; Id.,30:2407-2412, 1983). Antibodies have been developed againstschistosomular surface antigens and have been found to act againstschistosomulae in vivo or in vitro (Simpson et al., Parasitology,83:163-177, 1981; Smith et al., Parasitology, 84:83-91, 1982: Gryzch etal., J. Immunol., 129:2739-2743, 1982; Zodda et al., J. Immunol.129:2326-2328, 1982; Dissous et al., J. immunol., 129:2232-2234, 1982)

Trypanosoma cruzi is the causative agent of Chagas' disease, and istransmitted by blood-sucking reduviid insects. An antibody has beengenerated that specifically inhibits the differentiation of one form ofthe parasite to another (epimastigote to trypomastigote stage) in vitro,and which reacts with a cell-surface glycoprotein; however, this antigenis absent from the mammalian (bloodstream) forms of the parasite (Sheret al., Nature, 300:639-640, 1982).

Anti-fungal antibodies are known in the art, such as anti-Sclerotiniaantibody (U.S. Pat. No. 7,910,702); antiglucuronoxylomannan antibody(Zhong and Priofski, 1998, Clin Diag Lab Immunol 5:58-64); anti-Candidaantibodies (Matthews and Burnie, 2001, 2:472-76); andanti-glycosphingolipid antibodies (Toledo et al., 2010, BMC Microbiol10:47).

Suitable antibodies have been developed against most of themicroorganism (bacteria, viruses, protozoa, fungi, other parasites)responsible for the majority of infections in humans, and many have beenused previously for in vitro diagnostic purposes. These antibodies, andnewer antibodies that can be generated by conventional methods, areappropriate for use in the present invention.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, scFv and the like. Otherantibody fragments include, but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression libraries can be constructed (Huse et al., 1989, Science,246:1274-1281) to allow rapid and easy identification of monoclonal Fab′fragments with the desired specificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredisclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

An antibody fragment can be prepared by known methods, for example, asdisclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

A single complementarity-determining region (CDR) is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. (See, e.g., Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780).

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;Hwang and Foote, 2005, Methods 36:3-10; Clark, 2000, Immunol Today21:397-402; J Immunol 1976 117:1056-60; Ellison et al., 1982, Nucl AcidsRes 13:4071-79; Stickler et al., 2011, Genes and Immunity 12:213-21).

Multispecific and Multivalent Antibodies

Methods for producing bispecific antibodies include engineeredrecombinant antibodies which have additional cysteine residues so thatthey crosslink more strongly than the more common immunoglobulinisotypes. (See, e.g., FitzGerald et al, Protein Eng. 10(10):1221-1225,(1997)). Another approach is to engineer recombinant fusion proteinslinking two or more different single-chain antibody or antibody fragmentsegments with the needed dual specificities. (See, e.g., Coloma et al.,Nature Biotech. 15:159-163, (1997)). A variety of bispecific antibodiescan be produced using molecular engineering. In one form, the bispecificantibody may consist of, for example, an scFv with a single binding sitefor one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bispecific antibody may consist of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen. In alternative embodiments,multispecific and/or multivalent antibodies may be produced using thedock-and-lock (DNL) technique as described below.

In certain embodiments, the bispecific antibody may bind to twodifferent antigens, such as antigens selected from the group consistingof CD19, CD20, CD22, CD74, CD79a, CD40L, ILGF-R1, TROP2, CEACAM5,CECAM6, HLA-DR, IFNα, IL-6 and TNF-α. In other embodiments, of use inpretargeting methods described in more detail below, the bispecificantibody may contain at least one binding site for a disease associatedantigen, such as a tumor-associated antigen (TAA) and at least onebinding site for a hapten on a targetable construct.

Dock-and-Lock (DNL)

In preferred embodiments, bispecific or multispecific antibodies orother constructs may be produced using the dock-and-lock technology(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787;7,666,400; 7,858,070; 7,871,622; 7,906,121; 7,906,118 and 7,901,680, theExamples section of each incorporated herein by reference). The DNLmethod exploits specific protein/protein interactions that occur betweenthe regulatory (R) subunits of cAMP-dependent protein kinase (PKA) andthe anchoring domain (AD) of A-kinase anchoring proteins (AKAPs)(Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat.Rev. Mol. Cell Biol. 2004; 5: 959). PKA, which plays a central role inone of the best studied signal transduction pathways triggered by thebinding of the second messenger cAMP to the R subunits, was firstisolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol.Chem. 1968; 243:3763). The structure of the holoenzyme consists of twocatalytic subunits held in an inactive form by the R subunits (Taylor,J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two typesof R subunits (RI and RII), and each type has α and β isoforms (Scott,Pharmacol. Ther. 1991; 50:123). Thus, the four types of PKA regulatorysubunit are RIα, RIβ, RIIα and RIIβ. The R subunits have been isolatedonly as stable dimers and the dimerization domain has been shown toconsist of the first 44 amino-terminal residues (Newlon et al., Nat.Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads tothe release of active catalytic subunits for a broad spectrum ofserine/threonine kinase activities, which are oriented toward selectedsubstrates through the compartmentalization of PKA via its docking withAKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIα,RIβ, RIIα or RIIβ and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chimura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL constructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118 and 7,901,680.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct) that iscleared within minutes from the blood. A pre-targeting bispecific ormultispecific antibody, which has binding sites for the targetableconstruct as well as a target antigen, is administered first, freeantibody is allowed to clear from circulation and then the targetableconstruct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Targetable Constructs

In certain embodiments, targetable construct peptides labeled with oneor more therapeutic or diagnostic agents for use in pre-targeting may beselected to bind to a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used in a pretargeting technique wherein the antibodymay be administered first to a subject. Sufficient time may be allowedfor the bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂(SEQ ID NO:41), wherein DOTA is1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and HSG is thehistamine succinyl glycyl group. Alternatively, DOTA may be replaced byNOTA (1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties. Chelating moieties may be used,for example, to bind to a therapeutic and or diagnostic radionuclide,paramagnetic ion or contrast agent.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity. Exemplary methods of peptidesynthesis are disclosed in the Examples below.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

Preparation of Immunoconjugates

In preferred embodiments, a therapeutic or diagnostic agent may becovalently attached to an antibody or antibody fragment to form animmunoconjugate. Where the immunoconjugate is to be administered inconcentrated form by subcutaneous, intramuscular or transdermaldelivery, the skilled artisan will realize that only non-cytotoxicagents may be conjugated to the antibody. Where a second antibody orfragment thereof is administered by a different route, such asintravenously, either before, simultaneously with or after thesubcutaneous, intramuscular or transdermal delivery, then the type ofdiagnostic or therapeutic agent that may be conjugated to the secondantibody or fragment thereof is not so limited, and may comprise anydiagnostic or therapeutic agent known in the art, including cytotoxicagents.

In some embodiments, a diagnostic and/or therapeutic agent may beattached to an antibody or fragment thereof via a carrier moiety.Carrier moieties may be attached, for example to reduced SH groupsand/or to carbohydrate side chains. A carrier moiety can be attached atthe hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

Therapeutic and Diagnostic Agents

In certain embodiments, the antibodies or fragments thereof may be usedin combination with one or more therapeutic and/or diagnostic agents.Where the agent is attached to an antibody or fragment thereof to beadministered by subcutaneous, intramuscular or transdermaladministration of a concentrated antibody formulation, then onlynon-cytotoxic agents are contemplated. Non-cytotoxic agents may include,without limitation, immunomodulators, cytokines (and their inhibitors),chemokines (and their inhibitors), tyrosine kinase inhibitors, growthfactors, hormones and certain enzymes (i.e., those that do not inducelocal necrosis), or their inhibitors. Where the agent is co-administeredeither before, simultaneously with or after the subcutaneous,intramuscular or transdermal antibody formulation, then cytotoxic agentsmay be utilized. An agent may be administered as an immunoconjugate witha second antibody or fragment thereof, or may be administered as a freeagent. The following discussion applies to both cytotoxic andnon-cytotoxic agents.

Therapeutic agents may be selected from the group consisting of aradionuclide, an immunomodulator, an anti-angiogenic agent, a cytokine,a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme,an oligonucleotide, a pro-apoptotic agent, an interference RNA, aphotoactive therapeutic agent, a tyrosine kinase inhibitor, asphingosine inhibitor, a cytotoxic agent, which may be achemotherapeutic agent or a toxin, and a combination thereof. The drugsof use may possess a pharmaceutical property selected from the groupconsisting of antimitotic, antikinase, alkylating, antimetabolite,antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents, andcombinations thereof.

Exemplary drugs may include, but are not limited to, 5-fluorouracil,aplidin, azaribine, anastrozole, anthracyclines, bendamustine,bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, epipodophyllotoxin, estrogen receptor binding agents,etoposide (VP16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracilmustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.

Toxins may include ricin, abrin, alpha toxin, saporin, ribonuclease(RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.

Immunomodulators may be selected from a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and acombination thereof. Specifically useful are lymphotoxins such as tumornecrosis factor (TNF), hematopoietic factors, such as interleukin (IL),colony stimulating factor, such as granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF),interferon, such as interferons-α, -β or -γ, and stem cell growthfactor, such as that designated “S1 factor”. Included among thecytokines are growth hormones such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;prostaglandin, fibroblast growth factor; prolactin; placental lactogen,OB protein; tumor necrosis factor-α and -β; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-β; platelet-growth factor; transforminggrowth factors (TGFs) such as TGF-α and TGF-β; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, and -γ; colony stimulating factors(CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs) such as IL-1,IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25,LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumornecrosis factor and LT.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Radioactive isotopes include, but are not limited to—¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay-energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²⁵⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-placenta growth factor (PlGF)peptides and antibodies, anti-vascular growth factor antibodies (such asanti-VEGF and anti-PlGF), anti-Flk-1 antibodies, anti-Flt-1 antibodiesand peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF(macrophage migration-inhibitory factor) antibodies, laminin peptides,fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16Kprolactin fragment, Linomide, thalidomide, pentoxifylline, genistein,TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline maybe of use.

The therapeutic agent may comprise an oligonucleotide, such as a siRNA.The skilled artisan will realize that any siRNA or interference RNAspecies may be attached to an antibody or fragment thereof for deliveryto a targeted tissue. Many siRNA species against a wide variety oftargets are known in the art, and any such known siRNA may be utilizedin the claimed methods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (7,781,575), and apolipoproteinB (7,795,421), the Examples section of each referenced patentincorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either a siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Exemplary siRNA species known in the art are listed in Table 2. AlthoughsiRNA is delivered as a double-stranded molecule, for simplicity onlythe sense strand sequences are shown in Table 2.

TABLE 2 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 1 VEGF R2 AAGCTCAGCACACAGAAAGACSEQ ID NO: 2 CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO: 3 CXCR4GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 4 PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 5 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 6 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 7 E1 A binding proteinUGACACAGGCAGGCUUGACUU SEQ ID NO: 8 Plasminogen GGTGAAGAAGGGCGTCCAASEQ ID NO: 9 activator K-ras GATCCGTTGGAGCTGTTGGCGTAGTTCAA SEQ ID NO: 10GAGACTCGCCAACAGCTCCAACTTTTGGA AA Sortilin 1 AGGTGGTGTTAACAGCAGAGSEQ ID NO: 11 Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 12Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 13 Bcl-XUAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 14 Raf-1TTTGAATATCTGTGCTGAGAACACAGTTC SEQ ID NO: 15 TCAGCACAGATATTCTTTTTHeat shock AATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID NO: 16transcription factor 2 IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 17Thioredoxin AUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 18 CD44GAACGAAUCCUGAAGACAUCU SEQ ID NO: 19 MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTCSEQ ID NO: 20 MAPKAPK2 UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 21 FGFR1AAGTCGGACGCAACAGAGAAA SEQ ID NO: 22 ERBB2 CUACCUUUCUACGGACGUGdTdTSEQ ID NO: 23 BCL2L1 CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 24 ABL1TTAUUCCUUCUUCGGGAAGUC SEQ ID NO: 25 CEACAM1 AACCTTCTGGAACCCGCCCACSEQ ID NO: 26 CD9 GAGCATCTTCGAGCAAGAA SEQ ID NO: 27 CD151CATGTGGCACCGTTTGCCT SEQ ID NO: 28 Caspase 8 AACTACCAGAAAGGTATACCTSEQ ID NO: 29 BRCA1 UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 30 p53GCAUGAACCGGAGGCCCAUTT SEQ ID NO: 31 CEACAM6 CCGGACAGTTCCATGTATASEQ ID NO: 32

The skilled artisan will realize that Table 2 represents a very smallsampling of the total number of siRNA species known in the art, and thatany such known siRNA may be utilized in the claimed methods andcompositions.

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹⁸F,⁵²Fe, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y,⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br,⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds. A widevariety of fluorescent labels are known in the art, including but notlimited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Methods of Administration

The subject antibodies and immunoglobulins in general may be formulatedto obtain compositions that include one or more pharmaceuticallysuitable excipients, surfactants, polyols, buffers, salts, amino acids,or additional ingredients, or some combination of these. This can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients (i.e., the labeled molecules)are combined in a mixture with one or more pharmaceutically suitableexcipients. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients are wellknown to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parenteral injection, more preferably by subcutaneous,intramuscular or transdermal delivery. Other forms of parenteraladministration include intravenous, intraarterial, intralymphatic,intrathecal, intraocular, intracerebral, or intracavitary injection. Inparenteral administration, the compositions will be formulated in a unitdosage injectable form such as a solution, suspension or emulsion, inassociation with a pharmaceutically acceptable excipient. Suchexcipients are inherently nontoxic and nontherapeutic. Examples of suchexcipients are saline, Ringer's solution, dextrose solution and Hanks'solution. Nonaqueous excipients such as fixed oils and ethyl oleate mayalso be used. An alternative excipient is 5% dextrose in saline. Theexcipient may contain minor amounts of additives such as substances thatenhance isotonicity and chemical stability, including buffers andpreservatives.

Formulated compositions comprising antibodies can be used forsubcutaneous, intramuscular or transdermal administration. Compositionscan be presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. Compositions can also take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the compositionscan be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The compositions may be administered in solution. The formulationthereof should be in a solution having a suitable pharmaceuticallyacceptable buffer such as phosphate, TRIS (hydroxymethyl)aminomethane-HCl or citrate and the like. Buffer concentrations shouldbe in the range of 1 to 100 mM. The formulated solution may also containa salt, such as sodium chloride or potassium chloride in a concentrationof 50 to 150 mM. An effective amount of a stabilizing agent such asmannitol, trehalose, sorbitol, glycerol, albumin, a globulin, adetergent, a gelatin, a protamine or a salt of protamine may also beincluded.

The dosage of an administered antibody for humans will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibody that is inthe range of from about 1 mg to 600 mg as a single infusion, although alower or higher dosage also may be administered. Typically, it isdesirable to provide the recipient with a dosage that is in the range offrom about 50 mg per square meter (m²) of body surface area or 70 to 85mg of the antibody for the typical adult, although a lower or higherdosage also may be administered. Examples of dosages of antibodies thatmay be administered to a human subject are 1 to 1,000 mg, morepreferably 1 to 70 mg, most preferably 1 to 20 mg, although higher orlower doses may be used. Dosages may be repeated as needed, for example,once per week for 4-10 weeks, preferably once per week for 8 weeks, andmore preferably, once per week for 4 weeks. It may also be given lessfrequently, such as every other week for several months.

More recently, subcutaneous administration of veltuzumab has been givento NHL patients in 4 doses of 80, 160 or 320 mg, repeated every twoweeks (Negrea et al., 2011, Haematologica 96:567-73). Only occasional,mild to moderate and transient injection reactions were observed, withno other safety issues (Id.). The objective response rate (CR+CRu+PR)was 47%, with a CR/CRu (complete response) rate of 24% (Id.).Interestingly, the 80 mg dosage group showed the highest percentage ofobjective response (⅔, 67%), with one of three patients showing acomplete response (Id.). Four out of eight objective responses continuedfor 60 weeks (Id.). All serum samples evaluated for HAHA were negative(Id.). Although the low sample population reported in this studyprecludes any definitive conclusions on optimal dosing, it is apparentthat therapeutic response was observed at the lowest dosage tested (80mg).

In certain alternative embodiments, the antibody may be administered bytransdermal delivery. Different methods of transdermal delivery areknown in the art, such as by transdermal patches or by microneedledevices, and any such known method may be utilized. In an exemplaryembodiment, transdermal delivery may utilize a delivery device such asthe 3M hollow Microstructured Transdermal System (hMTS) for antibodybased therapeutics. The hMTS device comprises a 1 cm² microneedle arrayconsisting of 18 hollow microneedles that are 950 microns in length,which penetrate approximately 600-700 microns into the dermal layer ofthe skin where there is a high density of lymphatic channels. Aspring-loaded device forces the antibody composition from a fluidreservoir through the microneedles for delivery to the subject. Onlytransient erythema and edema at the injection site are observed (Burtonet al., 2011, Pharm Res 28:31-40). The hMTS device is not perceived as aneedle injector, resulting in improved patient compliance.

In alternative embodiments, transdermal delivery of peptides andproteins may be achieved by (1) coadminstering with a synthetic peptidecomprising the amino acid sequence of ACSSSPSKHCG (SEQ ID NO:42) asreported by Chen et al. (Nat Biotechnol 2006; 24: 455-460) andCarmichael et al. (Pain 2010; 149:316-324); (2) coadministering witharginine-rich intracellular delivery peptides as reported by Wang et al.(BBRC 2006; 346: 758-767); (3) coadminstering with either AT1002(FCIGRLCG, SEQ ID NO:43) or Tat (GRKKRRNRRRCG, SEQ ID NO:44) as reportedby Uchida et al. (Chem Pharm Bull 2011; 59:196); or (4) using anadhesive transdermal patch as reported by Jurynczyk et al (Ann Neurol2010; 68:593-601). In addition, transdermal delivery of negativelycharged drugs may be facilitated by combining with the positivelycharged, pore-forming magainin peptide as reported by Kim et al. (Int JPharm 2008; 362:20-28).

In preferred embodiments where the antibody is administeredsubcutaneously, intramuscularly or transdermally in a concentratedformulation, the volume of administration is preferably limited to 3 mlor less, more preferably 2 ml or less, more preferably 1 ml or less. Theuse of concentrated antibody formulations allowing low volumesubcutaneous, intramuscular or transdermal administration is preferredto the use of more dilute antibody formulations that require specializeddevices and ingredients (e.g., hyaluronidase) for subcutaneousadministration of larger volumes of fluid, such as 10 ml or more. Thesubcutaneous, intramuscular or transdermal delivery may be administeredas a single administration to one skin site or alternatively may berepeated one or more times, or even given to more than one skin site inone therapeutic dosing session. However, the more concentrated theformulation, the lower the volume injected and the fewer injections willbe needed for each therapeutic dosing.

Methods of Use

In preferred embodiments, the concentrated antibodies are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioma, melanoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers are noted belowand include: squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, endometrial cancer or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, anal carcinoma, penile carcinoma, as well as head andneck cancer. The term “cancer” includes primary malignant cells ortumors (e.g., those whose cells have not migrated to sites in thesubject's body other than the site of the original malignancy or tumor)and secondary malignant cells or tumors (e.g., those arising frommetastasis, the migration of malignant cells or tumor cells to secondarysites that are different from the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma,Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult PrimaryLiver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma,AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer,Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, BreastCancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System(Primary) Lymphoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, ChildhoodBrain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood CerebralAstrocytoma, Childhood Extracranial Germ Cell Tumors, ChildhoodHodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamicand Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, ChildhoodMedulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal andSupratentorial Primitive Neuroectodermal Tumors, Childhood Primary LiverCancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,Childhood Visual Pathway and Hypothalamic Glioma, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-CellLymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma andRelated Tumors, Exocrine Pancreatic Cancer, Extracranial Germ CellTumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, EyeCancer, Female Breast Cancer, Gallbladder Cancer, Gastric Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ CellTumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head andNeck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin'sLymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, IntestinalCancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet CellPancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer,Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used todetect or treat malignant or premalignant conditions. Such uses areindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia,pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be detected include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be detected and/or treatedinclude, but are not limited to, benign dysproliferative disorders(e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia,keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solarkeratosis.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

The exemplary conditions listed above that may be treated are notlimiting. The skilled artisan will be aware that antibodies or antibodyfragments are known for a wide variety of conditions, such as autoimmunedisease, graft-versus-host-disease, organ transplant rejection,cardiovascular disease, neurodegenerative disease, metabolic disease,cancer, infectious disease and hyperproliferative disease.

Exemplary autoimmune diseases include acute idiopathic thrombocytopenicpurpura, chronic immune thrombocytopenia, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,pemphigus vulgaris, juvenile diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis and fibrosing alveolitis.

Infectious diseases may be caused by a variety of pathogenic organisms,such as bacteria, viruses or mycoplasma. Exemplary known infectiousagents include, but are not limited to, Streptococcus agalactiae,Legionella pneumophilia, Streptococcus pyogenes, Esherichia coli,Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilusinfluenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonasaeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacteriumtuberculosis, HIV-1, HIV-2, HIV-3, Hepatitis A, Hepatitis B, HepatitisC, Hepatitis D, Rabies virus, Influenza virus, Cytomegalovirus, Herpessimplex I and II, Human serum parvo-like virus, Respiratory syncytialvirus, Varicella-Zoster virus, Hepatitis B virus, Measles virus,Adenovirus, Human T-cell leukemia viruses, Epstein-Barr virus, Mumpsvirus, Vesicular stomatitis virus, Sindbis virus, Lymphocyticchoriomeningitis virus, Wart virus, Blue tongue virus, Sendai virus, Reovirus, Polio virus, Dengue virus, Rubella virus, Plasmodium falciparum,Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosomacruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumonia.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one concentrated antibody or fragment thereof as described herein.A device capable of delivering the kit components by injection, forexample, a syringe for subcutaneous injection, may be included. Wheretransdermal administration is used, a delivery device such as hollowmicroneedle delivery device may be included in the kit. Exemplarytransdermal delivery devices are known in the art, such as 3M's hollowMicrostructured Transdermal System (hMTS), and any such known device maybe used.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents.Alternatively, the concentrated antibody may be delivered and stored asa liquid formulation. Other containers that may be used include, but arenot limited to, a pouch, tray, box, tube, or the like. Kit componentsmay be packaged and maintained sterilely within the containers. Anothercomponent that can be included is instructions to a person using a kitfor its use.

EXAMPLES Example 1. Purification of hLL2 Anti-CD22 Antibody

The hLL2 anti-CD22 antibody (epratuzumab) was designed, constructed,cloned and transfected into myeloma host cells as described in U.S. Pat.Nos. 5,789,554 and 6,187,287, the Examples section of each of which isincorporated herein by reference. Use of appropriate leader sequencesresults in secretion of the antibody into the serum-free cell culturemedium. Cells may be removed by centrifugation and the antibody purifiedfrom culture medium as shown, for example, in FIG. 1.

Generally, the purification process for hLL2 IgG and other antibodiesdescribed in the following Examples features chromatography on threesequential columns of Protein A, Q-SEPHAROSE® and SP-SEPHAROSE®.Although SEPHAROSE® is used as an exemplary column chromatography resin,the skilled artisan will realize that alternative methods ofchromatography and alternative chromatography resins are known in theart and may be used. Further, the anion and cation exchange steps arenot limited to Q-SEPHAROSE® and SP-SEPHAROSE®, but may also utilizeother anion- and cation-exchange resins known in the art. The last stepof the process utilizes a DV20 virus removal filtration, after which theproduct is tested for sterility.

The Protein A affinity resin used for the first column, MAB SELECT™ (GEHealthcare, Piscataway, N.J.) has a binding capacity of 25-30 mg/mL. Theresin was packed up to a 20 cm height in a 40 cm diameter column to apacked bed volume of 25 L, with a maximum loading capacity of 625 gm.Before the antibody containing culture medium was loaded, the packedcolumn was sanitized with 0.1 M acetic acid in 20% ethanol and thenre-generated with 0.04 M PBS, pH 7.4. After equilibration, thesupernatant was loaded at a maximum flow rate of 300 cm/hr. The columnwas washed with 0.04 M PBS, pH 7.4, until the absorbance returned tobaseline, followed by washing with another 5 bed volumes of 0.04 M PBS,pH 7.4 at 300 cm/hr.

The bound IgG was eluted with 0.1 M citrate, pH 3.5, at a maximum flowrate of 300 cm/hr. The elution profile was monitored by absorbance at280 nm, using a flow through spectrophotometer. The collected productpeak was neutralized to pH 7.0-8.0 using 3 M Tris/HCl, pH 8.6. As anadditional virus removal step, the neutralized product peak was titratedto pH 3.5-3.7 using 1 M citric acid. This mixture was incubated at roomtemperature for four hours and at the end of the incubation, it wasneutralized to pH 7.0-8.0 using 3 M Tris/HCl, pH 8.6.

The mixture was then concentrated to 5-7 mg/mL and diafiltered into 0.02M Tris/HCl, 0.01M NaCl, pH 8.2, in preparation for the next purificationstep. The diafiltered Protein A purified hLL2 IgG was filtered through a0.2 μm filter and stored at 2-8° C. until further purification.

The anion exchange resin used for the next column was Q-SEPHAROSE® fastflow resin (GE Healthcare, Piscataway, N.J.). The resin was packed up toa 20 cm height in a 40 cm diameter column, to a packed bed volume of 25L with a maximum loading capacity of 625 gm. Before the Protein Apurified IgG was loaded, the packed column was sanitized with 1 M sodiumhydroxide and then regenerated with 0.02 M Tris/HCl, 1.0 M NaCl, pH 8.0.The resin was then equilibrated using 0.02 M Tris/HCl, 0.01M NaCl, pH8.2. The diafiltered Protein A purified IgG was loaded at a flow rate of100 cm/hr and the flow through peak was eluted with 0.02 M Tris/HCl,0.01M NaCl, pH 8.2 at a maximum flow rate of 300 cm/hr. The contaminantseluted from the Protein A column bound to the Q-SEPHAROSE® resin. TheQ-SEPHAROSE® purified IgG was filtered using a 0.2-μm filter and storedat 2-8° C. until further purification. Before loading onto the finalcolumn, the IgG was titrated to pH 5.0 using 1 M citric acid.

The cation exchange resin used for the last column was SP-SEPHAROSE®fast flow resin (GE Healthcare, Piscataway, N.J.). The resin was packedup to a 20 cm height in a 40 cm diameter column, with a maximum loadingcapacity of 625 gm. Before the Q-SEPHAROSE® purified hLL2 IgG wasloaded, the packed column was sanitized with 1 M sodium hydroxide andthen equilibrated with 0.025 M citrate, pH 5.0. The IgG was loaded at amaximum flow rate of 300 cm/hr and the column was washed with 5 bedvolumes of 0.025 M citrate, pH 5.0, at 300 cm/hr. The bound IgG peak wasthen eluted with 0.025 M citrate, 0.15 M sodium chloride, pH 6.0, at amaximum flow rate of 300 cm/hr. The elution profile was monitored byabsorbance at 280 nm.

The purified hLL2 IgG was filtered using a 0.2 μm filter and stored at2-8° C. before DV₂₀ filtration. The IgG was concentrated to 9.5-10.5mg/mL and then diafiltered into 0.04 M PBS, 0.075% Polysorbate 80, pH7.4. The IgG was then filtered through a 0.2 μm filter into a sterilecontainer, then filtered through a 0.1 μm filter into a sterile pressurevessel, then filtered through a 20 nm filter for virus removal.

Example 2. Purification of hLL1 Anti-CD74 Antibody

The hLL1 anti-CD74 antibody (milatuzumab) was designed, constructed,cloned and transfected into myeloma host cells as described in U.S. Pat.Nos. 7,312,318; 7,772,373; 7,919,087 and 7,931,903, the Examples sectionof each of which is incorporated herein by reference.

The hLL1 antibody was purified by essentially the same protocoldescribed in Example 1 above, with the following differences. TheProtein A resin was packed to a 20 cm height in a 20 cm diameter column,providing a packed bed volume of 6.3 L. The maximum loading capacity ofthe Protein A column was 220 gm. The Q-SEPHAROSE® column was packed to a20 cm height in a 30 cm diameter column to a packed bed volume of 14.1L, with a maximum loading capacity of 300 gm. The SP-SEPHAROSE® columnwas packed to a 20 cm height in a 20 cm diameter column, with a packedbed volume of 6.3 L and a maximum loading capacity of 220 gm. Thepurified hLL1 IgG was concentrated to 10-11 mg/mL for DV₂₀ filtration.After filtration, 75 mL of 0.04 M PBS, 1% Polysorbate 80, pH 7.4 wasadded to every liter of purified IgG and the mixture was filtered againthrough a 0.2 μm filter before storage at 2°-8° C.

Example 3. Purification of hL243 Anti-HLA-DR Antibody

The hL243 anti-HLA-DR antibody was designed, constructed, cloned andtransfected into myeloma host cells as described in U.S. Pat. No.7,612,180, the Examples section of which is incorporated herein byreference.

The hL243 antibody (IMMU-114) was purified by essentially the sameprotocol described in Example 1 above, with the following differences.The Protein A resin was packed to a 20 cm height in a 20 cm diametercolumn, providing a packed bed volume of 6.3 L. The maximum loadingcapacity of the Protein A column was 220 gm. The Protein A purified IgGwas concentrated to 5-7 mg/ml and diafiltered into 0.02 M Tris/HCl, 0.05M NaCl, pH 7.5, before filtration and loading onto the Q-SEPHAROSE®column.

The Q-SEPHAROSE® column was packed to a 20 cm height in a 30 cm diametercolumn to a packed bed volume of 14.1 L, with a maximum loading capacityof 300 gm. After sanitization with 1 M sodium hydroxide, the resin wasequilibrated with 0.02 M Tris/HCl, 0.05 M NaCl, pH 7.5. The flow throughpeak was eluted with 0.02 M Tris/HCl, 0.05 M NaCl, pH 7.5.

The SP-SEPHAROSE® column was packed to a 20 cm height in a 20 cmdiameter column, with a packed bed volume of 6.3 L and a maximum loadingcapacity of 220 gm. After loading and washing, the IgG was eluted with0.025 M citrate, 0.15 M NaCl, pH 6.0.

The purified hL243 IgG was concentrated to 10-11 mg/mL and diafilteredinto 0.04 M PBS, pH 7.4, then filtered through 0.2 and 0.1 μm filtersbefore DV₂₀ filtration. After filtration, 75 mL of 0.04 M PBS, 1%Polysorbate 80, pH 7.4 was added to every liter of purified IgG and themixture was filtered again through a 0.2 μm filter before storage at2°-8° C.

Example 4. Isotypes of hL243 Anti-HLA-DR Antibody

The hL243 anti-HLA-DR antibody was prepared as described in Example 3above. An expression vector hL243pdHL2 was constructed by sequentiallysubcloning the XbaI-BamHI and XhoI/NotI fragments of hL243V_(K) andV_(H), respectively, into pdHL2 as described previously (Losman et al.,1997. Cancer, 80:2660). The expression vector pdHL2, as described byGilles et al. (1989, J. Immunol. Methods 125:191), contains the genomicsequence of the human γ1 chain, therefore, the hL243 is an IgG1/Kisotype.

To construct the expression vector for hL243 of other isotypes, such asIgG4/K, the sequence of the human γ1 chain was replaced with that of thehuman γ4 chain, which was obtained by PCR amplification. The templateused was genomic DNA extracted from the ATCC CRL-11397 cell and theprimer pair was as below.

SacII (SEQ ID NO: 35) CCGCGGTCACATGGCACCACCTCTCTTGCAGCTTCCACCAAGGGCCCEagI (SEQ ID NO: 36) CCGGCCGTCGCACTCATTTACCCAGAGACAGGG

The amplified PCR product was cloned into TOPO® TA sequencing vector(INVITROGEN®) and the sequence was confirmed by DNA sequencing.

A point mutation, Ser241Pro (based on Kabat numbering) was introducedinto the hinge region of the γ4 sequence to avoid formation ofhalf-molecules when the IgG4 antibody is expressed in mammalian cellcultures (Schuurman et al., 2001, Mol. Immunol. 38:1). The human γ4hinge region sequence between PstI and StuI restriction sites (56 bp)was replaced with a synthetic DNA fragment with substitution of the TCAcodon for Ser241 to CCG for Pro. The human γ1 sequence in hL243pdHL2 wassubstituted with the mutated γ4 sequence, resulting in the finalexpression vector, designated as hL243γ4PpdHL2, for the IgG4 isotypehL243. The IgG4 isotype was used in subsequent experiments ontherapeutic efficacy.

The amino acid sequences of the hL243 IgG4P heavy chain and of the hL243light chain are shown in FIG. 8.

Example 5. Purification of hA20 Anti-CD20 Antibody

The hA20 anti-CD20 antibody (veltuzumab) was designed, constructed,cloned and transfected into myeloma host cells as described in U.S. Pat.Nos. 7,151,164 and 7,435,803, the Examples section of which isincorporated herein by reference.

The hA20 antibody was purified by essentially the same protocoldescribed in Example 1 above, with the following differences. Protein Aresin with a maximum loading capacity of 30 mg/mL was packed to a 19-21cm height in a 20 cm diameter column, providing a packed bed volume of6.0-6.6 L. The maximum loading capacity of the Protein A column wasapproximately 180 gm. The Protein A purified IgG was titrated to pH3.6-3.8 with 1 M citric acid for virus removal.

The Q-SEPHAROSE® column was packed to a height of 19-21 cm in a 30 cmdiameter column to a packed bed volume of 13.4-14.9 L, with a maximumloading capacity of 300 gm. After loading, the flow-through peak waseluted with 0.2 M Tris/HCl, 0.01 M NaCl, pH 8.0.

The SP-SEPHAROSE® column was packed to a 19-21 cm height in a 20 cmdiameter column, with a packed bed volume of 6.0-6.6 L and a maximumloading capacity of 180 gm. After loading and washing, the IgG waseluted with 0.025 M citrate, 0.15 M NaCl, pH 6.0.

The purified hA20 IgG was concentrated to 10-11 mg/mL and diafilteredinto 0.04 M PBS, pH 7.4, then filtered through 0.2 and 0.1 μm filtersbefore DV₂₀ filtration. After filtration, 75 mL of 0.04 M PBS, 1%Polysorbate 80, pH 7.4 was added to every liter of purified IgG and themixture was filtered again through a 0.2 μm filter before storage at2°-8° C.

Example 6. Ultrafiltration Concentration of Humanized Antibodies in HighConcentration Formulation Buffer

Using ultrafiltration, humanized IgG was concentrated to at least 200mg/mL in High Concentration Formulation (HCF) buffer, with minimal or noaggregation. A series of analytical assays were performed to monitor anychanges during the concentration process. No detectable changes inantibody quality or solution characteristics were observed. The liquidformulation was stable at 2-8° C. for at least 12 months. The stabilityestimated at 12 months by SE-HPLC (which showed essentially a singlepeak on the absorbance trace, FIG. 4-6) was between 97 and 99% (Table4). Reducing and non-reducing PAGE was consistent with the HPLC results(FIG. 2A-2B). The formulation is suitable for subcutaneous injection(SC). Exemplary antibodies tested include milatuzumab (hLL1, anti-CD74),epratuzumab (hLL2, anti-CD22), veltuzumab (hA20, anti-CD20) and hL243(anti-HLA-DR; IMMU-114).

A High Concentration Formulation (HCF) buffer was developed that wasdemonstrated to be capable of stabilizing antibody solutions to at least200 mg/mL concentration (Table 3). In addition to phosphate buffer andNaCl from IV formulation, this SC formulation contains mannitol whichhas been of use in protein formulations for maintaining stability andisotonicity, and Polysorbate 80 (PS-80) which protects antibodiesagainst aggregation. Since the pI value of most humanized IgG1antibodies is between 8-9.5, a citric acid/sodium citrate buffer system(buffering range 2.5˜5.6) and a low pH (5.2) were used to ensure theprotein is in charged form, and thus more stable in solution.

During ultrafiltration a 50 KD MW cut-off membrane was used, whichretained and concentrated the 150 KD IgG molecules while allowing waterand small molecules in the formulation buffer to pass through.

TABLE 3 High Concentration Formulation Compositions hLL1 hLL2 hA20 hL243(Milatuzumab, (Epratuzumab, (Veltuzumab, (anti-HLA- Component anti-CD74)anti-CD22) anti-CD20) DR) IgG₁ 213 mg/mL 109 mg/mL 162 mg/ml 101 mg/mLNa₂HPO₄•7H₂O 2.30 g NaH₂PO₄•H₂O 0.76 g Sodium Chloride 6.16 gPolysorbate 80 1.0 mL (w/v) (polysobate-80 was added at the end of theconcentration step) Sodium Citrate 0.34 g Dihydrate Citric Acid 1.3 gMonohydrate Mannitol 12.0 g WFI (qs) 1 L pH (adjusted by NaOH) 5.2

The solute concentrations of HCF buffer were 6.2 mM citric acidmonohydrate, 105 mM sodium chloride, 1.2 mM sodium citrate dihydrate,8.7 mM sodium phosphate dibasic, 5.5 mM sodium phosphate monobasic, 66mM mannitol, pH 5.2, conductivity 11.0-14.0 mS/cm.

An AMICON® Model 8050 Stirred Ultrafiltration Cell (from MILLIPORE®, 50mL max volume) was used with a 50 KD polyethersulfone filter NMWL (fromMILLIPORE®, diameter 44.5 mm) to concentrate the antibodies. Ultra pureargon gas was used to pressurize the system.

The UF-cell with a 50 KD membrane was assembled and connected to theargon gas supply. The cell was rinsed and filled with buffer. With thestirrer on, pressure was applied to run more than two volumes of HCFbuffer through the membrane. From this point on, the membrane wasmaintained in a wet state.

After rinsing of the stirred cell chamber, the residual buffer wasdiscarded and the cell was filled with IgG solution. The stir plate wasthen started and the pressure applied. The antibody solution wasconcentrated to approximately one half (½) the original volume, thendiafiltered using HCF buffer (5× retentate volume). The process wasrepeated 3-4 times until the diafiltration was completed and checked tomake sure that the pH and conductivity of filtrate was identical to theHCF buffer.

Post-concentration, Polysobate-80 was added so that the finalconcentration of Polysorbate was 0.1%. The IgG was then filtered througha 0.22-μm filter, placed in clear glass vials, and stored at 2-8° C.until analytical testing was performed.

Each sample was visually inspected against a dark background under lightfor any particulates and precipitates. IgG protein concentration wasmeasured by UV (OD₂₈₀) absorbance after serial dilutions. SDS-PAGE wasperformed using pre-cast 4-20% gradient gels. Ten μL of ˜1 mg/mL samplewas heated at 95° C. for 3 minutes in the presence (reducing gel) orabsence (non-reducing gel) of a 3% 2-mercaptoethanol solution. Gels werestained with 0.1% Coomassie Blue. Isoelectric Focusing (IEF) wasperformed by standard techniques, using pH 6-10.5 gradient gels. Sampleswere diluted to 2 mg/mL and applied at 5 μL each along with pI markersand reference standard. Gels were stained with Coomassie Blue andscanned for quantification of pI range.

Size Exclusion HPLC (SE-HPLC) was carried out using a BECKMAN® HPLCsystem (Model 116), with a BIO-SIL® SEC 250 column. The sample wasdiluted to about 1 mg/mL and 60 μL was injected. The elution buffer wascomposed of 0.05 M NaH₂PO₄, 0.05 M Na₂HPO₄ and 1 mM EDTA, pH 6.8. Theelution was monitored by UV absorbance at 280 nm.

All analytical results are summarized in Table 4. The SDS-PAGE gel (FIG.2A non-reducing and FIG. 2B, reducing), IEF gel (FIG. 3), and SE-HPLCchromatograms (FIG. 4 to FIG. 6) are shown. It can be seen thatultrafiltration concentration of the IgG in HCF buffer from 101 mg/mL to213 mg/mL did not result in any detectable changes in the purified IgG.

TABLE 4 Analytical Results Antibody hLL1 hLL2 hA20 hL243 Concentration213 mg/mL 109 mg/mL 102 mg/mL 101 mg/mL SE-HPLC 98.3% 98.5% 98.9% 99.3%(Area Percent) (0 month)  (0 Month)  (0 Month)  (0 Month) 97.5% 97.3%98.5% 98.8% (4 month) (12 Month) (12 Month) (12 Month) Visual ClearClear Clear Clear inspection yellowish yellowish yellowish slight milkcolor color color color SDS-Page gel Reducing and Non-Reducing SDS-PAGEgels for all samples of concentrated MAb showed a band pattern similarto reference standard IEF gel IEF gel patterns for all samples ofconcentrated MAb showed a band pattern similar to reference standard

This study demonstrated that in the HCF buffer, IgG could beconcentrated by ultrafiltration up to 213 mg/mL without any visibleaggregation or precipitation. Other quality aspects of the antibody suchas molecular integrity, charge variation and solution pH were alsomaintained.

Example 7. High-Protein Concentration Antibody Formulations forSubcutaneous or Intramuscular Injection

Alternative high concentration formulations for subcutaneous orintramuscular administration may comprise amino acids, such as arginineor glutamine. A comparison of the maximal protein concentrationachievable without precipitation was determined for epratuzumab(humanized anti-CD22), using three different formulations comprising thesugar mannitol and/or the amino acids arginine and glutamic acid (Table5).

Epratuzumab was applied to a 40 mL MABSELECT® (Protein A) affinitychromatography column, which was washed with phosphate-buffered salineand then diH₂O, to remove polysorbate-80 from the original bulkmaterial. The antibody was eluted with 80 mL of 0.05 M sodium citrate,pH 3.5. The eluate was neutralized by the addition of 132 mL of 0.1 MNaH₂PO₄ and formulated into CPREM buffer by the addition of 60 mL of a 1M L-arginine monohydrochloride/1 M L-glutamic acid (monosodium salt)solution and 39.6 mL of 1 M mannitol, adjusted to pH 5.3 with HCl anddiluted to 600 mL with deionized H₂O. The final CPREM formulationcontained 66 mM mannitol, 100 mM arginine, 100 mM glutamic acid, 144 mMNa, 100 mM Cl, 7.3 mM citrate, 22 mM phosphate, pH 5.3. A proteinconcentration of 2.56 mg/mL was measured by UV spectrophotometry at 280nM (OD₂₈₀).

The 600 mL solution was concentrated 120-fold using a stir-cellconcentrator with a 50 kDa MWCO membrane. A protein concentration of 238mg/mL in the 120× concentrate was measured by OD₂₈₀. There was noevident precipitation by visual inspection and an SE-HPLC trace, whichwas indistinguishable from that of the pre-concentration material,showed no evidence of aggregation (data not shown). The 120-foldconcentrate was separated into three aliquots.

An aliquot (0.5 mL) of the 120× concentrate (238 mg/mL) was maintainedin the CPREM formulation and further concentrated to 170× (0.35 mL) andmeasured by OD₂₈₀ at a protein concentration of 298 mg/mL withoutevident precipitation. SE-HPLC analysis resolved an identical trace tothe pre-concentration material with no aggregation (data not shown).Further concentration of the 30% protein solution was not attempted dueto high viscosity and limiting volumes.

A second aliquot was diafiltered into CPRE buffer (100 mM arginine, 100mM glutamic acid, 144 mM Na, 100 mM Cl, 7.3 mM citrate, 22 mM phosphate,pH 5.3.), which is CPREM buffer without mannitol. The CPRE proteinsolution was concentrated until a precipitate was evident. At thispoint, concentration was terminated and the solution was filtered. Theprotein concentration in the filtered concentrate was measured at 99mg/mL by OD₂₈₀.

The third aliquot was diafiltered into CPM buffer (66 mM mannitol, 144mM Na, 100 mM Cl, 7.3 mM citrate, 22 mM phosphate, pH 5.3.), which isCPREM without arginine and glutamic acid. The CPM protein solution wasconcentrated until a precipitate was evident. At this point,concentration was terminated and the solution was filtered. The proteinconcentration in the filtered concentrate was measured at 137 mg/mL byOD₂₈₀.

These results suggest that addition of arginine and glutamic acid to theHCF buffer of Example 6 increased the maximum concentration of antibodythat could be maintained without precipitation, up to at least 300mg/ml. Further, since maximum concentration of the hLL1 antibody thatcould be obtained in HCF buffer was no higher than observed with theother tested antibodies, and substantially lower than observed with thehLL1 antibody in HCF buffer (Table 4), it is expected that comparableincreases in stable antibody concentration without precipitation may beobtained for other highly concentrated antibodies.

TABLE 5 High-concentration epratuzumab formulations Arginine GlutamicMannitol C_(max) Formulation (mM) Acid (mM) (mM) (mg/L) CPREM 100 100 66298^(‡ ) CPRE 100 100 0  99* CPM 0 0 66 137* Each formulation contained144 mM Na, 100 mM Cl, 7.3 mM citrate, 22 mM PO₄, pH 5.3 C^(max), maximalachievable concentration at the point of protein precipitation^(‡) orlimiting viscosity*

Example 8. Subcutaneous Injection of Low-Dose Veltuzumab inNon-Hodgkin's Lymphoma (NHL)

Veltuzumab was prepared for subcutaneous administration as described inExamples 5 and 6 above. Seventeen patients with previously untreated orrelapsed NHL received 4 doses of 80, 160 or 320 mg veltuzumab injecteds.c. every two weeks (Negrea et al., 2011, Haematologica 96:567-573).Responses were assessed by CT scans, with other evaluations includingadverse event, B-cell blood levels, serum veltuzumab levels and humananti-veltuzumab (HAHA) titers.

Only occasional, mild to moderate transient injection reactions wereseen with the s.c. injection and no other safety issues were observed.The s.c. veltuzumab exhibited a slow release pattern over several days,with mean maximum serum concentrations of 19, 25 and 64 μg/mL at dosagesof 80, 160 or 320 mg per injection. Transient B-cell depletion wasobserved at all dosage levels of veltuzumab. The objective response rate(partial responses plus complete responses plus complete responsesunconfirmed) was 47% ( 8/17) with a complete response/complete responseunconfirmed rate of 24% ( 4/17). Four of the eight objective responsescontinued for 60 weeks or more. Objective responses were observed at alldose levels of s.c. veltuzumab. All serum samples evaluated for humananti-veltuzumab antibody (HAHA) were negative.

It was concluded that subcutaneous injections of low-dose veltuzumab areconvenient, well-tolerated and capable of achieving sustained serumlevels, B-cell depletion and durable objective responses in indolentnon-Hodgkin's lymphoma.

Example 9. Subcutaneous Injection of Low-Dose Veltuzumab in ImmuneThrombocytopenic Purpura (ITP)

Eleven adult chronic ITP patients with platelet counts below 30×10⁹ andwho had failed at least one standard therapy received 2 doses of 80 or120 mg veltuzumab administered two weeks apart, either intravenously(n=7) or subcutaneously (n=4). Of the 9 evaluable patients, the overallobjective response rate was 67%, with 33% of patients having a completeresponse. For the subgroup of 6 patients who did not undergo surgicalspleen removal prior to the study, the response rate was 100%,regardless of the route of administration and across the two dosestested. More importantly, 50% of the subgroup completely responded toveltuzumab and continued to maintain their platelet levels at 6 weeks, 6months and 9 months post therapy. For the 3 patients who had undergonesplenectomy, none responded to treatment. Both s.c. and i.v. veltuzumabresulted in B-cell depletion. One patient had an infusion reaction toi.v. veltuzumab and discontinued treatment. Two other patients had minorimmunogenic responses to i.v. veltuzumab. No other safety issues wereobserved and no patients receiving s.c. veltuzumab exhibited animmunogenic response.

This study demonstrated the convenience, safety and efficacy ofveltuzumab for ITP therapy and the superiority of s.c. veltuzumab forreducing immunogenic response to administration of the antibody.

Example 10. Subcutaneous Injection of Low-Dose Epratuzumab in ChronicLymphocytic Leukemia (CLL)

Patients with previously untreated or relapsed CLL receive 4 doses of80, 160 or 320 mg epratuzumab injected s.c. every week or every twoweeks. Divided doses can also be administered twice weekly. Onlyoccasional mild to moderate transient injection reactions are seen withthe s.c. injection and no other safety issues are observed. The s.c.epratuzumab exhibits a slow release pattern over several days. TransientB-cell depletion is observed at all dosage levels of epratuzumab, butmore strikingly at the two highest doses given for at least 4 weeks.Objective responses are observed at all dose levels of s.c. epratuzumab,but with particularly high responses of 30% (mostly partial responses)at the highest dose. All serum samples evaluated for humananti-epratuzumab antibody (HAHA) are negative.

It is concluded that subcutaneous injections of low-dose epratuzumab areconvenient, well-tolerated and capable of achieving sustained serumlevels, B-cell depletion and durable objective responses in CLL.

Example 11. Subcutaneous Injection of Low-Dose Epratuzumab in SystemicLupus Erythematosus (SLE)

Epratuzumab is prepared for subcutaneous administration as described inExamples 1 and 5 or 6 above. Epratuzumab is prepared for subcutaneousadministration as described in Examples 1 and 5 or 6 above. Anopen-label, single-center study of 14 patients with moderately activeSLE (total British Isles Lupus Assessment Group (BILAG) score 6 to 12)is conducted. Patients receive 400 mg epratuzumab subcutaneously everyweek for 6 weeks. Evaluations include safety, SLE activity (BILAG),blood levels of B and T-cells and human anti-epratuzumab antibody (HAHA)titers. Total BILAG scores decrease by at least 50% in all 14 patients,with 92% having decreases continuing to at least 18 weeks. Almost allpatients (93%) experience improvement in at least one BILAG B- orC-level disease activity at 6, 10 and 18 weeks. Additionally, 3 patientswith multiple BILAG B involvement at baseline have completely resolvedall B-level disease activities by 18 weeks. Epratuzumab is welltolerated, with no evidence of immunogenicity or significant changes inT cells, immunoglobulins or autoantibody levels. B-cell levels decreaseby an average of 35% at 18 weeks and remain depressed for 6 monthspost-treatment. These results demonstrate the safety and efficacy ofsubcutaneous epratuzumab for treatment of SLE.

Example 12. Subcutaneous Injection of Milatuzumab in Multiple Myeloma

Milatuzumab is prepared for subcutaneous administration as described inExamples 1 and 6 above. Patients with relapsed multiple myeloma who hadfailed at least two standard therapies receive 10 doses of 300 mgmilatuzumab, injected s.c. at weekly intervals, of the naked antibody.Responses are classified by EBMT criteria, with PK and immunogenicityevaluated by serum milatuzumab levels and human anti-milatuzumabantibody (HAHA) titers, respectively. Only occasional mild to moderatetransient injection-site reactions are seen with the s.c. injection andno other safety issues are observed. The s.c. milatuzumab exhibits aslow release pattern over several days. Objective responses are observedat this dose level of s.c. milatuzumab, as measured by decrease of serumlight chains, IgM, circulating and marrow myeloma cells, and improvementin the patient's platelet, hemoglobin, and WBC levels due to improvedbone marrow function. All serum samples evaluated for humananti-milatuzumab antibody (HAHA) are negative.

Combination therapy of s.c. naked milatuzumab with bortezomib,doxorubicin or dexamethasone is observed to improve response in multiplemyeloma patients, as shown in preclinical models (Stein et al., 2009,Clin Cancer Res 15:2808-17). The combination therapy of milatuzumab withbortezomib, doxorubicin or dexamethasone produces a therapeutic effectthat is greater than that observed with milatuzumab alone, drug alone,or the combined effect of antibody or drug administered alone. Thecombination results in a significant reduction in the optimal dosesrequired of the drugs.

It is concluded that subcutaneous injections of milatuzumab areconvenient, well-tolerated and capable of achieving sustained serumlevels and durable objective responses in multiple myeloma.

Example 13. Allotype Conversion from G1 ml to nG1m1 LowersImmunogenicity of IgG1 Therapeutic Antibodies

Adalimumab, an anti-TNFα antibody, was constructed from a fully humanmonoclonal antibody with a G1m17,1 allotype. Adalimumab is approved fortherapy of various autoimmune diseases, such as rheumatoid arthritis,psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasisand juvenile idiopathic arthritis. Adalimumab is known to induce ananti-globulin response in some adalimumab-treated patients andantibodies against adalimumab are associated with non-response totreatment (Bartelds et al., 2010, Arthritis Res Ther 12:R221).

Adalimumab is transformed by site-directed mutagenesis from a G1m17,1allotype to a G1m3 allotype by making the heavy chain substitutionsK214R, D356E and L358M, resulting in G1m3-adalimumab.

The native adalimumab and G1m3-adalimumab are administered to patientsthat are homozygous for G1m17,1, heterozygous for G1m17,1 and G1m3 orhomozygous for G1m3. The allotype engineered G1m3-adalimumab is observedto induce a lower rate of immune response in the recipient, with fewerimmune reactions that require termination of administration.Surprisingly, the change to G1m3-adalimumab results in a largerreduction in immune response in G1m17,1 homozygotes than in G1m3homozygotes.

These results demonstrate that changing antibody allotype from G1m1 tonG1m1 can result in a reduction in immunogenicity of therapeuticantibodies.

Example 14. Transdermal Administration of Therapeutic Antibodies

A 3M hollow Microstructured Transdermal System (hMTS) is used to delivera concentrated antibody composition by transdermal administration(Burton et al., 2011, Pharm Res 28:31-40). Up to 1.5 mL of antibodyformulation at a concentration of between 100 to 300 mg/mL isadministered at a rate of up to 0.25 mL/min. A red blotch, the size ofthe hMTS array, is observed immediately after patch removal but is fadedas to be almost indistinguishable 10 min post-patch removal. Apart fromthis transient localized reaction, no other adverse infusion-relatedreaction is observed. Either whole antibodies or antibody fragments areadministered. Antibodies administered by transdermal delivery includeveltuzumab, milatuzumab, epratuzumab, adalimumab and clivatuzumab.Therapeutic efficacy and P_(K) profiles are similar to those observedwith subcutaneous administration of the same antibodies.

Veltuzumab is prepared for subcutaneous administration as described inExamples 5 and 6 above. Patients with previously untreated or relapsedNHL receive 4 doses of 80, 160 or 320 mg veltuzumab injectedtransdermally using a 3M hMTS device every two weeks (Negrea et al.,2011, Haematologica 96:567-573; Burton et al., 2011, Pharm Res28:31-40). Responses are assessed by CT scans, with other evaluationsincluding adverse event, B-cell blood levels, serum veltuzumab levelsand human anti-veltuzumab (HAHA) titers.

Only occasional, mild to moderate transient injection reactions wereseen with the transdermal injection and no other safety issues wereobserved. The transdermal veltuzumab exhibits a slow release patternover several days. Transient B-cell depletion is observed at all dosagelevels of veltuzumab. The objective response rate (partial responsesplus complete responses plus complete responses unconfirmed) is 45% witha complete response/complete response unconfirmed rate of 25%. Half ofthe objective responses continue for 60 weeks or more. Objectiveresponses are observed at all dose levels of transdermal veltuzumab. Allserum samples evaluated for human anti-veltuzumab antibody (HAHA) arenegative.

It is concluded that transdermal administration of low-dose veltuzumabis convenient, well-tolerated and capable of achieving sustained serumlevels, B-cell depletion and durable objective responses in indolentnon-Hodgkin's lymphoma.

Example 15. Subcutaneous Injection of Bispecific Antibody forPre-targeting

Anti-TROP2 Bispecific Antibody

TROP2 (also known as epithelial glycoprotein-1 or EGP-1) is apancarcinoma marker that is expressed at high levels on virtually allprostate carcinomas (PC). The bispecific antibody TF12(anti-TROP2×anti-HSG) is made by the dock-and-lock technique, asdescribed in Paragraphs 0077-0081 above, using the hRS7 and 679antibodies. TF12 effectively targets PC, and provides good tumor uptake.The efficacy of pretargeted radioimmunotherapy (PRIT) using TF12 and¹⁷⁷Lu-labeled diHSG-peptide (IMP288) is studied in mice with s.c. PC3tumors.

Five groups of athymic mice (n=10) are xenografted with s.c. human PC3tumors. Mice are injected s.c. with 2.5 nmol of TF12 followed 16 h laterby i.v. injection of 0.1 nmol of radiolabeled IMP288. The therapeuticefficacy of one or two cycles (48 h apart) PRIT with TF12/¹⁷⁷Lu-IMP288(41 MBq) is compared to that of conventional RIT with ¹⁷⁷Lu-labeledanti-TROP2 mAb at the MTD (11 MBq ¹⁷⁷Lu-hRS7). Control groups receiveeither PBS or ¹⁷⁷Lu-IMP288 (41 MBq) without pretargeting with TF12.

Mice treated with one cycle of PRIT show improved median survival (121vs. 82 days). Two cycles of PRIT significantly enhance median survivalcompared to only one cycle (>160 vs. 121 days, p<0.0001). Therapeuticefficacy of two cycles of PRIT does not differ significantly from RITwith ¹⁷⁷Lu-hRS7 (p=0.067), but mice treated with PRIT show lowerhematologic toxicity. Decrease in leukocytes is significantly higherwith RIT than observed with PRIT (67% vs. 38%, p=0.028). After 150 days,70% of the mice treated with two cycles of PRIT and all mice treatedwith conventional RIT are still alive, compared to none in the controlgroups.

Pretargeting with TF12 in combination with ¹⁷⁷Lu-IMP288 is an excellentsystem for specific radioimmunotherapy of prostate cancer. Efficacy isenhanced significantly by adding an extra cycle of PRIT. In view of thelimited toxicity there is room for additional treatment cycles or higherdoses of ¹⁷⁷Lu-IMP288.

Anti-CD20 Bispecific Antibody

CD20 is a tumor-associated antibody (TAA) that is a significant targetfor antibody-based therapy in a wide variety of hematologic cancers andautoimmune diseases. The TF4 bispecific antibody comprising hA20(anti-CD20)×h679 (anti-HSG) was made according to Sharkey et al. (2008,Cancer Res 68:5282-90).

The TF4 bispecific antibody is administered by s.c. injection to nudemice with Ramos B cell lymphomas. Ramos cells (1×10⁷) are implanted s.c.in 6- to 8-wk old female BALB/c nude mice 2 to 4 weeks prior to antibodyadministration. TF4 antibody in amounts ranging from 0.125 to 1.0 nmolare administered, followed at 29 h later by ⁹⁰Y-labeled IMP 288 (Sharkeyet al., 2008, Cancer Res 68:5282-90). Optimal results are obtained witha 20-fold ration of TF4 to targetable construct peptide.

The TF4 bispecific antibody is cleared rapidly from the blood, withprocessing in liver, spleen and kidney. At 24 hours, TF4 improves tumoruptake of labeled peptide 2.6 fold and enhances tumor to blood ratio45-fold compared to an anti-CD20 Fab×anti-HSG Fab chemical conjugate andby 1.6-fold and 1,600-fold, respectively, compared to radiolabeledanti-CD20 IgG. A severe (>90%) and prolonged reduction of WBCs isobserved at the maximum dose of ⁹⁰Y-labeled anti-CD20 IgG, whilepretargeting results in <60% reduction. TF4 pretargeting results inhighly significant improvement in survival, curing 33% to 90% of animalseven at relatively low doses, whereas most tumors progress rapidly whentreated with ⁹⁰Y-labeled anti-CD20 IgG.

Example 16. Administration of Bispecific Antibody for Cancer Therapy

Anti-CD20×Anti-CD22 Bispecific Antibody

The CD20 and CD22 antigens are widely expressed in cancers ofhematopoietic origin. Combination therapy with anti-CD20 and anti-CD22antibodies has been demonstrated to be more efficacious than eitheragent alone and to be effective for therapy of cancers resistant tosingle agent treatments (e.g., Leonard et al., 2005, J Clin Oncol23:5044-51; Qu et al., 2008, Blood 111:2211-19; Gupta et al., 2010,Blood 116:3258-67). Using the techniques disclosed herein, therapy withdifferent antibodies may be administered as combinations of theindividual antibodies, or as bispecific antibody constructs.

A bispecific construct comprising the anti-CD20 antibody veltuzumab andthe anti-CD22 antibody epratuzumab are prepared by the dock-and-lock(DNL) technique as previously described (U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; the Examples section of each of which incorporatedherein by reference). Concentrated solutions of bispecific DNL constructof between 100 and 300 mg/ml are prepared as described in Examples 6 and7 above. The concentrated bispecific DNL construct is administered topatients with recurrent B-cell lymphoma by s.c. injection at dosagesranging from 200 to 600 mg per injection, once a week for four weeks.Patients are selected with indolent histologies (including follicularlymphoma), aggressive NHL and DLBCL.

The treatment is well tolerated with minimal infusion-related transientreactions. Over 60% of patients with follicular NHL achieve objectiveresponse (OR), with a over 50% complete responses (CRs). Over 60% ofpatients with DLBCL achieve OR with 50% CRs. Median time to progressionfor all indolent NHL patients is 17.8 months.

It is concluded that therapy with an anti-CD22×anti-CD20 bispecificantibody is efficacious for treatment of hematopoietic tumors, withimproved efficacy compared to single antibody treatment and decreasedtoxicity.

Anti-CD20×Anti-CD74 Bispecific Antibody

A bispecific DNL antibody construct comprising veltuzumab (anti-CD20)and milatuzumab (anti-CD74) is prepared as previously described (Guptaet al., 2012, Blood 119:3767-78). A concentrated formulation of thebispecific anti-CD20×anti-CD74 DNL construct is prepared as described inExamples 6 and 7 above and is administered by s.c. injection to subjectswith mantle cell lymphoma (MCL) and other lymphomias or leukemias.

The juxtaposition of CD20 and CD74 on MCL cells by the bispecificantibody results in homotypic adhesion and triggers intracellularchanges that include loss of mitochondrial transmembrane potential,production of reactive oxygen species, rapid and sustainedphosphorylation of ERKs and JNK, down-regulation of pAkt and Bcl-xL andlysosomal membrane permeabilization, resulting in cell death. Thebispecific antibody significantly extends the survival of nude micebearing MCL xenografts in a dose-dependent manner. Other lymphoma andleukemia cell lines are also depleted by exposure to the bispecificantibody. The results demonstrate the in vitro and in vivo efficacy ofthe anti-CD20×anti-CD74 bispecific antibody construct for therapy ofhematopoietic tumors.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated herein by reference,including any Tables and Figures, to the same extent as if eachreference had been incorporated by reference individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the invention.

What is claimed is:
 1. A method of subcutaneous, intramuscular ortransdermal administration of a therapeutic antibody or immunoglobulincomprising: a) obtaining a composition comprising the antibody orimmunoglobulin at a concentration of at least 225 mg/ml, wherein thecomposition further comprises citric acid monohydrate, sodium chloride,sodium citrate dihydrate, sodium phosphate dibasic, sodium phosphatemonobasic, polysorbate 80 and mannitol at a pH of 4.5 to 5.5 and whereinthe antibody is selected from the group consisting of hR1 (anti-IGF-1R),hPAM4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31(anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243(anti-HLA-DR), hL243 IgG4P (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15(anti-CEACAM6), hRS7 (anti-EGP-1), hMN-3 (anti-CEACAM6), and hRFB4(anti-CD22); and b) administering the composition to a patient bysubcutaneous, intramuscular or transdermal delivery, wherein the volumeof the composition administered is selected from the group consisting of3 ml or less, 2 ml or less and 1 ml or less.
 2. The method of claim 1,wherein the composition comprises 6.2 mM citric acid monohydrate, 105 mMsodium chloride, 1.2 mM sodium citrate dihydrate, 8.7 mM sodiumphosphate dibasic, 5.5 mM sodium phosphate monobasic, 0.1% polysorbate80 and 66 mM mannitol.
 3. The method of claim 1, wherein the antibody orimmunoglobulin is concentrated to at least 250 mg/ml or at least 300mg/ml.
 4. The method of claim 1, wherein the amount of antibody orimmunoglobulin administered is selected from the group consisting of 40mg, 80 mg, 160 mg, 240 mg and 320 mg.
 5. The method of claim 1, whereinthe administration is repeated.
 6. The method of claim 1, wherein thesubcutaneously, intramuscularly or transdermally administered antibodyis effective at a lower dose than the same antibody administeredintravenously.
 7. The method of claim 1, wherein the pH of thecomposition is 5.2.
 8. The method of claim 1, wherein the compositionfurther comprises arginine and glutamic acid.
 9. The method of claim 1,wherein the antibody has glutamate at Kabat residue 356 and methionineat Kabat residue 358 of the antibody heavy chain.
 10. The method ofclaim 9, wherein the antibody has arginine at Kabat residue 214 of theantibody heavy chain.
 11. The method of claim 9, wherein the antibodyhas glutamate at Kabat residue 356, methionine at Kabat residue 358 andalanine at Kabat residue 431 of the antibody heavy chain.
 12. The methodof claim 9, wherein the antibody has alanine at Kabat residue 153 andvaline at Kabat residue 191 of the antibody light chain.
 13. The methodof claim 9, wherein the antibody comprises heavy chain constant regionamino acid residues arginine-214, glutamic acid-356, methionine-358 andalanine-431.
 14. The method of claim 1, wherein the antibody is selectedfrom the group consisting of a monoclonal antibody, an antigen-bindingfragment of a monoclonal antibody, a bispecific antibody, amultispecific antibody, an immunoconjugate and an antibody fusionprotein.
 15. The method of claim 1, wherein the immunoconjugatecomprises at least one non-cytotoxic therapeutic or diagnostic agent.16. The method of claim 15, wherein the therapeutic agent is selectedfrom the group consisting of an immunomodulator, a cytokine, achemokine, a tyrosine kinase inhibitor, a growth factor, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interleukin (IL), an interferon (IFN), ahormone and an enzyme.
 17. The method of claim 15, wherein thetherapeutic agent is selected from the group consisting oferythropoietin, thrombopoietin tumor necrosis factor-α (TNF), TNF-β,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, stem cell growth factor designated “S1factor”, human growth hormone, N-methionyl human growth hormone, bovinegrowth hormone, parathyroid hormone, thyroxine, insulin, proinsulin,relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), luteinizing hormone (LH), hepatic growthfactor, prostaglandin, fibroblast growth factor, prolactin, placentallactogen, OB protein, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,macrophage-CSF (M-CSF), IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin,thrombospondin, endostatin and LT.
 18. The method of claim 1, whereinthe antibody is a naked antibody.
 19. The method of claim 18, furthercomprising administering at least one therapeutic agent to saidindividual.
 20. The method of claim 19, wherein the therapeutic agent isselected from the group consisting of a drug, a prodrug, a toxin, anenzyme, a tyrosine kinase inhibitor, a sphingosine inhibitor, animmunomodulator, a cytokine, a hormone, a second antibody, a secondantibody fragment, an immunoconjugate, a radionuclide, an antisenseoligonucleotide, an RNAi, an anti-angiogenic agent, a pro-apoptosisagent and a cytotoxic agent.
 21. The method of claim 1, wherein theantibody is selected from the group consisting of epratuzumab,veltuzumab, milatuzumab, hL243 (anti-HLA-DR) and hL243 IgG4P(anti-HLA-DR).
 22. The method of claim 1, wherein the antibody isepratuzumab.
 23. The method of claim 1, wherein the antibody isveltuzumab.
 24. The method of claim 1, wherein the antibody ismilatuzumab.
 25. The method of claim 1, wherein the antibody isclivatuzumab.
 26. The method of claim 1, wherein the antibody islabetuzumab.
 27. The method of claim 1, wherein the antibody is hL243.28. The method of claim 1, wherein the patient has a disease selectedfrom the group consisting of autoimmune disease, immune dysregulationdisease, infectious disease, carcinoma, sarcoma, lymphoma, leukemia,chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-celllymphoma, multiple myeloma, non-Hodgkin's lymphoma, Alzheimer's disease,type-2 diabetes, type-1 diabetes, amyloidosis, cardiovascular disease,neurological disease and metabolic disease.
 29. The method of claim 28,wherein the autoimmune disease is selected from the group consisting ofacute idiopathic thrombocytopenic purpura, chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myastheniagravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis and fibrosing alveolitis.
 30. The methodof claim 14, wherein the antibody fragment is selected from the groupconsisting of F(ab′)₂, F(ab)₂, Fab, Fab′ and scFv fragments.
 31. Themethod of claim 14, wherein the bispecific antibody comprises at leastone binding site for a tumor-associated antigen and at least one bindingsite for a hapten and the method further comprises administering to thepatient a targetable construct, wherein the targetable constructcomprises at least one copy of the hapten and is attached to at leastone diagnostic or therapeutic agent.